Use of delta tocopherol for the treatment of lysosomal storage disorders

ABSTRACT

This disclosure relates generally to the treatment of lysosomal storage disorders. Specifically, the disclosure relates to a novel use of delta tocopherol in the treatment of diseases and conditions related to lysosomal storage disorders. Included in the present disclosure is a method for the modulation of cholesterol recycling. Further, the disclosure relates to conditions such as Niemann-Pick type C disease, Farber disease, Niemann-Pick type A disease, Wolman disease and Tay Sachs disease. Further included in the present disclosure is a method for treating lysosomal storage disorders comprising the administration of delta tocopherol. Further included in the present disclosure is a method for treating lysosomal storage disorders comprising the administration of delta tocopherol in combination with cyclodextrin to a patient in need thereof.

RELATED APPLICATIONS

This application claims priority to U.S. Application No. 61/365,712,which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This disclosure relates generally to the treatment of lysosomal storagedisorders. Specifically, the disclosure relates to a novel use of deltatocopherol in the treatment of diseases and conditions related tolysosomal storage disorders. Included in the present disclosure is amethod for the modulation of cholesterol recycling. Further, thedisclosure relates to conditions such as Niemann-Pick type C disease,Farber disease, Niemann-Pick type A disease, Wolman disease and TaySachs disease. Further included in the present disclosure is a methodfor treating lysosomal storage disorders comprising the use ofδ-tocopherol. Further included in the present disclosure is a method fortreating lysosomal storage disorders comprising the use of δ-tocopherolin combination with cyclodextrin.

BACKGROUND

Tocopherols are natural products widely used as vitamin E for humanhealth, cosmetic ingredients and food antioxidants. The main source oftocopherols is the deodorized distillate produced by the manufactureprocess of edible oil. The main constituents of the tocopherolsextracted from deodorized distillate are α, β, γ, and δ-tocopherols.Their molecular structures have a common core ring, differing only inthe number of attached methyl groups. Although the structures are quitesimilar, it is known in the art that α-tocopherol has the highestbiopotency among all the isomeric forms. Current research involvingtocopherols is focused on α-tocopherol, emphasizing α-tocopherol's useas an antioxidant.

In normal physiological cell function, lysosomal enzymes break downmacromolecules in cells. Normal metabolism allows the cells to removeexpended metabolites. Defects or deficiencies of lysosomal enzymes orother lysosomal components can result in the accumulation of undegradedmetabolites. These defects or deficiencies result in Lysosomal StorageDisorders (LSDs).

Cholesterol is an essential component of the cell membrane and plays animportant role in maintaining integrity and fluidity of the cellmembrane with additional lipids. The appropriate proportion and makeupof cholesterol in the cell membrane is important for regulation of cellsignaling, receptor function, ion permeability and endocytosis. Inaddition, cholesterol is the precursor molecule for synthesis of bileacids, Vitamin D and steroid hormones. Cholesterol concentration isregulated on two levels; in the body as a whole and intracellularly.Cholesterol is primarily synthesized in cells and transported in bloodby lipid binding proteins. While the mechanisms of cholesterol transportin blood and cholesterol synthesis in cells are well characterized, themany details of free cholesterol trafficking in cells remain unclear.Cholesterol enters cells through low-density lipoprotein (LDL) receptorsvia endocytosis or cholesterol is de novo synthesized inside cells. Theinternalized cholesterol esters are hydrolyzed by acid lipase to formfree cholesterols in late endosomes/lysosomes that are delivered to NPC1and NPC2 proteins for further processing. Cholesterol may be transportedto other proteins and/or to membrane microdomains such as lipid raftsand other vesicles. It is known that most of these free cholesterolsrecycle back to cell membrane; some of them efflux out of cells, andsome of them move to ER for storage after esterification.

Lysosomal storage diseases (lysosomal storage disorders) are a group of˜50 diseases with a common feature of lysosomal accumulation ofmacromolecules such as lipids or glycoproteins in patient cells. Thediseases are caused by inherited genetic mutations that result indeficiency of lysosomal enzyme or protein. Once the hydrolysis ortransport of macromolecules such as lipids and glycoproteins is reducedby a mutation of an enzyme or protein in lysosome, they are accumulatedwhich causes the enlargement of lysososomes as well as malfunction oflipids recycling and utilization in cells. Degeneration or death ofaffected cells occurs in certain tissues which are varied in differentlysosomal storage diseases. The hepatosplenomegaly and neuronaldegeneration are common features of many lysosomal storage diseases.

One identified LSD is Niemann-Pick type C disease (NPCD), an autosomalrecessive disease with an estimated incidence of 1:150,000. This diseaseis characterized by a lysosomal accumulation of unesterified cholesteroland other lipids in many cell types, probably due to impairment of theretrograde transport of lipids from the late endosomes and/or lysosomesto the plasma membrane or endoplasmic reticulum (ER). The clinicalmanifestation includes hepatosplenomegaly, vertical gaze palsy, andprogressive neurodegeneration characterized by cerebellar ataxia, bulbardysfunction, and variable degrees of cognitive decline. Most often, theonset of symptoms occurs in early childhood, leading to death within adecade. Two human genes have been identified for NPCD: mutations in NPC1are causative in nearly 95% of all NPC cases, while mutations in NPC2account for the rest. Over 230 mutations have been identified in theNPC1 gene, nearly ⅔ of which are missense mutations. NPC1 is ahighly-conserved integral membrane protein with 13 transmembranedomains, while the NPC2 gene encodes a small soluble protein. Bothproteins are located in late endosomes and lysosomes. Recent structureand biochemical studies have revealed that NPC1 and NPC2 bind tocholesterol in opposite orientations, leading to a working model ofthese two proteins: NPC2 captures the cholesterol liberated from LDL,and then transfers it to the NPC1 on the membrane for subsequenttrafficking out of the lysosomes. In NPCD patients, the cholesteroltrafficking out of late endosomes and lysosomes is blocked resulting inan accumulation of free cholesterols in late endosomes and lysosomes.This accumulation of cholesterol eventually affects the lysosomalhomeostasis, triggering also the accumulation of other lysosomalsubstrates such us glycosphingolipids. Moreover, glycosphingolipidsaccumulation in sphingolipid storage diseases (SLSD) as result of adefective lysosomal hydrolase or activator protein also affectscholesterol homeostasis elevating cellular cholesterol levels.

Currently, there is no cure for NPCD and all established therapies arefor relief of symptoms with limited efficacy. Clinical trials withMiglustat (N-butyldeoxynojirimycin, Zavesca®) are in progress withfavorable preliminary results. Miglustat is an iminosugar that inhibitsglucosylceramide synthase, an enzyme responsible for a series ofreactions that lead to the synthesis of most glycosphingolipids (GSL).This drug crosses the blood-brain barrier, reduces the substrateavailability for synthesizing GSL, and thus reduces GSL accumulation inthe brain. This substrate reduction effect seems to relieve the symptomsin NPC patients. Recently, several other compounds includingallopregnanolone, T0901317, curcumin and cyclodextrin have also beenreported to have beneficial effects on NPC cell or animal models. Thefull therapeutic benefits of these drugs still need to be evaluated.Despite these studies, an effective treatment for NPC patients is stillan unmet medical need.

Recent advances in technologies for high-throughput screening (HTS) havemade it possible to screen the cell-based NPC disease model againstcompound libraries to identify lead compounds for the new drugdevelopment. Two such attempts have been made which applied the filipincholesterol staining assay in NPC patient-derived skin fibroblasts.Though some active compounds were reported from these screens, none ofthem are useful for the further drug development. In addition, the drugdevelopment process usually takes an average of 8-12 years costing onthe order of hundreds of millions of dollars. The failure rate of drugdevelopment is very high due to unpredictable compound toxicity in humanand disproportional efficacy between human and model systems. Thepresent invention is directed toward overcoming the problems discussedabove.

SUMMARY OF THE EMBODIMENTS

We identify, in one embodiment, the use of δ-tocopherol for thetreatment of LSDs. Further, we identify the use of δ-tocopherol inreducing the accumulation of free cholesterols. Additionally we teachthe use of δ-tocopherol for the reduction of the size of enlargedlysosomes.

In one embodiment, we disclose a method for treating lysosomal storagedisorder comprising administering to a patient in need thereof apharmaceutically effective amount of a δ tocopherol. In one embodiment,we disclose a pharmaceutically effective amount of δ tocopherol of 50 uMplasma and/or tissue concentration. In one embodiment, we disclose apharmaceutically effective amount of δ tocopherol of less than 50 uM. Inone embodiment, we disclose a pharmaceutically effective amount of δtocopherol of less than 40 uM. In one embodiment, we disclose apharmaceutically effective amount of δ tocopherol of less than 30 uM. Inone embodiment, we disclose a pharmaceutically effective amount of δtocopherol of less than 20 uM. In one embodiment, we disclose apharmaceutically effective amount of δ tocopherol of less than 15 uM. Inone embodiment, we disclose a pharmaceutically effective amount of δtocopherol of less than 10 uM. In one embodiment, we disclose apharmaceutically effective amount of δ tocopherol of less than 7.8 uM.In one embodiment, we disclose a pharmaceutically effective amount of δtocopherol without significant cytotoxicity.

The disclosure further provides a method for treating a mammal having alysosomal storage disorder by administering a pharmaceutical compositioncomprising a dosage of a δ-tocopherol effective to treat the lysosomalstorage disorder; and a beta-cyclodextrin compound, in which the dosageof the δ-tocopherol compound is less than about 1000 IU/kg per day. Inparticular embodiments, the dosage of δ-tocopherol is less than 500IU/kg per day, less than 50 IU/kg per day, less than 5 IU/kg per day, orless than 0.5 IU/kg per day. In one embodiment of the methods oftreatment, the disclosure provides a method for treating a mammal havinga lysosomal storage disorder by administering a pharmaceuticalcomposition comprising a dosage of a δ-tocopherol compound, wherein thedosage of the δ-tocopherol compound is less than an accepted dose ofα-tocopherol. In one embodiment of the methods of treatment, thedisclosure provides a method for treating a mammal having a lysosomalstorage disorder by administering a pharmaceutical compositioncomprising a dosage of a δ-tocopherol compound, wherein the dosage ofthe 6 -tocopherol is less than about 1000 IU/kg per day. In particularembodiments, the dosage of δ-tocopherol is less than 500 IU/kg per day,less than 50 IU/kg per day, less than 5 IU/kg per day, or less than 0.5IU/kg per day.

We further disclose the treatment with δ-tocopherol, wherein thelysosomal storage disorder is selected from the group consisting ofNiemann-Pick Disease, Mucopolysaccharidoses disorder, and NeuronalCeroid Lipofuscinoses. We further disclose the treatment withδ-tocopherol, wherein the lysosomal storage disorder is selected fromthe group consisting of Niemann-Pick type C disease, Farber disease,Niemann-Pick type A disease, Wolman disease and Tay Sachs disease. Inone embodiment, the composition of δ tocopherol excludes α, β, and γtocopherol for the treatment of LDS. In one embodiment, the compositionof δ-tocopherol excludes α, β, and γ tocopherol for the treatment ofNPCD. In one embodiment the composition of δ tocopherol excludes any oneof α, β, and γ tocopherols, and/or any combination thereof, for thetreatment of LDS. In one embodiment, the composition of δ tocopherolcomprises cyclodextrin for the treatment of LDS. In one embodiment thecomposition of δ tocopherol excludes any one of α, β, and γ tocopherols,and/or any combination thereof, for the treatment of LDS, and comprisescyclodextrin for the treatment of LDS. In one embodiment, thecomposition of δ-tocopherol excludes α, β, and γ tocopherol for thetreatment of NPCD. We disclose a method for reducing cholesterolaccumulation in cells comprising administering a composition of δtocopherol. We further disclose a method for reducing cholesterolaccumulation in cells comprising administering a composition of δtocopherol which excludes any one of α, β, and γ tocopherol orcombinations thereof. We further disclose a method for treatment of adisease characterized by increased cholesterol accumulation wherein thecholesterol accumulation in cells is reduced by a method comprisingadministering a composition of δ tocopherol which excludes any one of α,β, and γ tocopherol or combinations thereof to a patient in need.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A) The influence of delta-tocopherol on free cholesterol (greenlines, right y-axis) and ATP level (red lines, left y-axis) in WT andNPC fibroblasts. On day 0 cells were seeded in 1536-well plates. On day1, 3, or 5, delta-tocopherol dissolved in DMSO was added to the cells atvarious final concentrations as indicated in the figure. On day 7, thecells were examined for free cholesterol and ATP levels by Amplex Redcholesterol assay and ATP-lite assay, respectively. Free cholesterol andATP levels of δ-tocopherol treated cells were normalized against thoseof DMSO treated cells of the same type and expressed as the percentageof DMSO treated cells. Error bars represented standard error of n=8values. B) Filipin staining of the NPC cells treated with δ-tocopherol.On day 0, cells were seeded on black clear-bottom 96-well plates at 500cells/well. On day 1, culture medium was replaced with medium containingdifferent concentrations of δ-tocopherol or matching DMSO. On day 4,media were replaced again with ones containing fresh compounds. On day6, filipin staining (blue) as well as CellMask staining (red) wasperformed as described in the method section. C) Lysotracker staining ofthe NPC cells treated with δ-tocopherol. Cells were treated similarly asin FIG. 1B and stained by Lysotracker Red and Hoechst as described inthe method session.

FIG. 2. A) Structures of vitamin E analogs. B) The influence ofdifferent forms of vitamin E on free cholesterol level of NPC cells. C)The influence of other vitamins and anti-oxidants on the freecholesterol level of NPC cells. D) The influence of various compounds incellular ATP level of NPC cells. E) Delta-tocopherol treatment did notchange the expression level of NPC1 and HMG-CoA reductase in NPCfibroblasts.

FIG. 3. Lysotracker staining of lysosomal storage disease cells treatedwith δ-tocopherol. Cells were treated similarly as in FIG. 1 and stainedby Lysotracker Red and Hoechst as described in the method session.

FIG. 4. Effects of δ-tocopherol and α-tocopherols on (A) freecholesterol, (B) cholesterol ester and (C) sphingomyeline in NPC cells.NPC or WT cells were treated with DMSO, 80 uM α-tocopherol or 40 uMδ-tocopherol for 5 days. Cell pellets were collected and extracted forlipids. FC EC and sphingomyeline levels were measured by GC/MS. (D)Effects of of δ-tocopherols and α-tocopherols on cholesterol efflux.

FIG. 5. A) Filipin staining of the WT or NPC MSC cells. B) NPC cellstreated with δ-tocopherol or DMSO were co-stained with filipin and theantibody against neuronal beta III tubulin. Cells were treated andstained as described in the method section.

FIG. 6. A model for mechanism of action for tocopherols.

FIG. 7. A model for mechanism of action for tocopherols.

FIG. 8. Lysotracker staining images from WT (wild type) control cellsare in the top panel and from Farber disease fibroblasts are in thebottom panel. Drugs (80 uM alpha-tocopherol, 40 uM delta-tocopherol and300 uM methyl-beta-cyclodextrin) were treated for four days in cellculture. 40× objectives.

FIG. 9. Lysotracker staining images from WT (wild type) control cellsare in the top panel and from Niemann Pick Type A disease (NPA)fibroblasts are in the bottom panel. Drugs (80 uM alpha-tocopherol, 40uM delta-tocopherol and 300 uM methyl-beta-cyclodextrin) were treatedfor four days in cell culture. 40X objectives.

FIG. 10. Lysotracker staining images from WT (wild type) control cellsare in the top panel and from Wolman6144 disease fibroblasts (from twopatients) are in the bottom panel. Drugs (80 uM alpha-tocopherol, 40 uMdelta-tocopherol and 300 uM methyl-beta-cyclodextrin) were treated forfour days in cell culture. 40× objectives.

FIG. 11. Lysotracker staining images from WT (wild type) control cellsare in the top panel and from Wolman11851 disease fibroblasts (from twopatients) are in the bottom panel. Drugs (80 uM alpha-tocopherol, 40 uMdelta-tocopherol and 300 uM methyl-beta-cyclodextrin) were treated forfour days in cell culture. 40× objectives.

FIG. 12. Lysotracker staining images from WT (wild type) control cellsare in the top panel and from Tay Sachs disease fibroblasts are in thebottom panel. Drugs (80 uM alpha-tocopherol, 40 uM delta-tocopherol and300 uM methyl-beta-cyclodextrin) were treated for four days in cellculture. 40× objectives.

FIG. 13. Lysotracker staining images from WT (wild type) control cellsare in the top panel and from Farber disease fibroblasts are in thebottom panel. Drugs (80 uM alpha-tocopherol, 40 uM delta-tocopherol and300 uM methyl-beta-cyclodextrin) were treated for four days in cellculture. 20× objectives.

FIG. 14. Lysotracker staining images from WT (wild type) control cellsare in the top panel and from Niemann Pick Type A disease (NPA)fibroblasts are in the bottom panel. Drugs (80 uM alpha-tocopherol, 40uM delta-tocopherol and 300 uM methyl-beta-cyclodextrin) were treatedfor four days in cell culture. 20× objectives.

FIG. 15. Lysotracker staining images from WT (wild type) control cellsare in the top panel and from Wolman6144 disease fibroblasts (from onepatient) are in the bottom panel. Drugs (80 uM alpha-tocopherol, 40 uMdelta-tocopherol and 300 uM methyl-beta-cyclodextrin) were treated forfour days in cell culture. 20× objectives.

FIG. 16. Lysotracker staining images from WT (wild type) control cellsare in the top panel and from Wolman11851 disease fibroblasts (from onepatient) are in the bottom panel. Drugs (80 uM alpha-tocopherol, 40 uMdelta-tocopherol and 300 uM methyl-beta-cyclodextrin) were treated forfour days in cell culture. 20× objectives.

FIG. 17. Lysotracker staining images from WT (wild type) control cellsare in the top panel and from Tay Sachs disease fibroblasts are in thebottom panel. Drugs (80 uM alpha-tocopherol, 40 uM delta-tocopherol and300 uM methyl-beta-cyclodextrin) were treated for four days in cellculture. 20× objectives.

FIG. 18. Control WT cells treated with 80 uM alpha-tocopherol and 40 uMdelta-tocopherol for four days. Top panel with red emission is for totallipids including the polar lipids such as phospholipids. Bottom panel iswith emission for non-polar lipids including cholesteryl ester andtriglycerides.

FIG. 19. Images of Nile red staining in Farber disease cells treatedwith 80 uM alpha-tocopherol and 40 uM delta-tocopherol for four days.Top panel with red emission is for total lipids including the polarlipids such as phospholipids. Bottom panel is with emission fornon-polar lipids including cholesteryl ester and triglycerides.

FIG. 20. Images of Nile red staining in NPA disease cells treated with80 uM alpha-tocopherol and 40 uM delta-tocopherol for four days. Toppanel with red emission is for total lipids including the polar lipidssuch as phospholipids. Bottom panel is with emission for non-polarlipids including cholesteryl ester and triglycerides.

FIG. 21. Images of Nile red staining in Wolman6144 disease cells (fromone patient) treated with 80 uM alpha-tocopherol and 40 uMdelta-tocopherol for four days. Top panel with red emission is for totallipids including the polar lipids such as phospholipids. Bottom panel iswith emission for non-polar lipids including cholesteryl ester andtriglycerides.

FIG. 22. Images of Nile red staining in Wolman11851 disease cells (fromone patient) treated with 80 uM alpha-tocopherol and 40 uMdelta-tocopherol for four days. Top panel with red emission is for totallipids including the polar lipids such as phospholipids. Bottom panel iswith emission for non-polar lipids including cholesteryl ester andtriglycerides.

FIG. 23. Images of Nile red staining in Tay Sachs disease cells treatedwith 80 uM alpha-tocopherol and 40 uM delta-tocopherol for four days.Top panel with red emission is for total lipids including the polarlipids such as phospholipids. Bottom panel is with emission fornon-polar lipids including cholesteryl ester and triglycerides.

FIG. 24. Combination therapy of vitamin E and cyclodextrin. In Wolmandisease cells, the combination sub-effective concentrations ofalpha-tocopherol (20 uM) or delta-tocopherol (10 uM) with 10 uM MBCD(methyl-beta-cyclodextrin) showed cholesterol reduction effect as thesole therapy with 40 uM delta-tocopherol and 80 uM alpha-tocopherol.MBCD at 0.3 mM showed the time dependent reduction on cholesterol levelin these cells. These results indicated the synergetic or additiveeffect for delta-tocopherol and clyclodextrin for the treatment oflysosomal storage diseases. This drug combination therapy has advantagesincluding reduced dosages for both drugs (to reduce potential adverseeffects) and enhanced therapeutic effect (because two drugs havedifferent mechanism of action).

Unless otherwise indicated, all numbers expressing quantities ofingredients, dimensions reaction conditions and so forth used in thespecification and claims are to be understood as being modified in allinstances by the term “about”.

In this application and the claims, the use of the singular includes theplural unless specifically stated otherwise. In addition, use of “or”means “and/or” unless stated otherwise. Moreover, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one unit unless specifically statedotherwise.

DETAILED DESCRIPTION

We disclose δ-tocopherol for the treatment of LSDs. Surprisingly,δ-tocopherol is the most potent of the eight isoforms of vitamin E forthe reduction of cholesterol accumulation in NPCD fibroblasts. Wedisclose a potency with delta tocopherol 6 to 8 times higher than withα-, β- and γ-tocopherols (EC₅₀ values between 60-80 μM). It has beenreported that in human plasma α-tocopherol is the most abundant vitaminE vitamin with a concentration in the range of 26 uM. The concentrationsfor β- and γ-tocopherols are much less (˜1-3 μM) whereas the plasmaconcentration for δ-tocopherol is the lowest at the tracer value (˜0.1μM). We disclose δ-tocopherol for the treatment of LSDs. δ-tocopherolhas a low physiological concentration in the body and is significantlypotent in reduction of cholesterol accumulation.

Vitamin E, as disclosed in the art, generally refers to α tocopherol;however, other vitamers or isoforms exist, which include α-, β-, γ- andδ-isoforms. The differences in α-, β-, γ- and δ-isoforms relies on theposition and number of methyl groups on the phenol ring, in addition tothe corresponding tocotrienols having three double bonds in the sidechain. The function of vitamin E is thought to be through itsantioxidant effects in cell membrane by reducing free radicals criticalfor the structural integrity of cells. As a lipid-soluble molecule,vitamin E is believed to insert into the cell membrane for its function.The plasma concentration of vitamin E is one per 2000-3000 lipids and iseasily depleted if it is not regenerated or is deficient in diet. Thedeficiency of vitamin E presents symptoms primarily in neuronal systemincluding ataxia, dysarthria, hyporeflexia and reduced vibration senseas well as cardiomyopathy and retinitis pigmentosa. On other hand, highdoses of vitamin E have been used and propose for the treatment ofneurological disorders. The potential link of vitamin E deficiency withcancer and inflammation has been reported with regard to the antioxidantscavenging activity of vitamin E.

Lysosomal storage diseases include many disorders such asmucopolysaccharidosis, mucolipidosis, spingolipidosis, GM2gangliosidosis, GM2 activator protein deficiency, Niemann-Pick disease,Gaucher's disease, Fabry's disease, Farber's disease, Metachromaticleukodystrophy, mucosulfatidosis, Krabbe's disease, Sphingolipidactivator protein deficiencies, Lysosomal acid lipase deficiency,Wolman's disease, Cholesterol ester storage disease, Cerebrotendiousxanthomatosis, Neuronal ceroid lipofuscinosis, oligosaccharidosis andrelated disorders, and lysosomal transport defects.

Blood cholesterol level of cholesterol is well regulated wherein ˜70%cholesterol is synthesized by the body and ˜30% of it isabsorbed/reabsorbed from the intestine. Cholesterol is a vitalstructural component in cell membrane, as cellular cholesterolhomeostasis is important to maintain a variety of cellular functions.Cholesterol is usually bound to lipid binding proteins in blood anddelivered to cells by LDL. The endocytosis formed coated vesiclescontaining LDL reach the late endosome/lysosme and the free cholesterolsare released from LDL by acid lipase. The free cholesterol is thendelivered to NPC2 and NPC1 proteins located in late endosome/lysosme.Free cholesterol is then moved to the trans-golgi network (TNG) forefflux through cell membrane and/or to ER for esterification for normalintracellular cholesterol storage. Mutations in NPC1/2 proteins resultin the malfunction in this cholesterol trafficking pathway in cells withthe consequence of free cholesterol accumulation in lateendosome/lysosme. This cholesterol recycling malfunction subsequentlyresults in the accumulation of other membrane lipids in the NPC cells.In addition, it is well known that cholesterol and sphingolipidsaccumulation induce oxidative stress. Thus, in the case of NPCoxysterols accumulation has been link with the apoptotic triggering. Inbrain, loss of Purkinje neurons in the cerebellar cortex due to neuronaldegeneration is most predominant in NPCD. Purkinje neurons rely on thecholesterols synthesized in astrocytes which are delivered to them.Deficiency in NPC1 or NPC2 proteins may block the cholesterol movementfrom late endosome/lysosme to other compartments in cells which impairsthe critical lipid recycling/movement (including many other lipids) andeventually results in cell death. We disclose a correction ofcholesterol movement malfunction in NPC cells with anactivation/enhancement of an alternative cholesterol recycling pathwayas a new therapeutic strategy to treat the deadly NPCD. In addition, theantioxidative effect of δ-tocopherol should have an impact in reducingthe oxysterols levels and therefore decreasing the apoptotic signal aspart of the overall proposed NPC treatment. Similar effects in reducingthe oxidative stress observed in other LSD should being considered.

We disclose tocopherols in NPC cells as well as in other LSD cells. Innormal conditions, cholesterol recycling/movement in the membrane mainlyrelies on the NPC1/NPC2 protein dependent pathway. The cholesterolinflux and efflux is balanced maintaining normal cellular functions.These functions include the rapid endocyctic/exocytic processes andlipid movement/recycling in the membrane system. The continuous lipidmovements in the plasma membrane and in intracellular compartments arecritical for maintaining the proper functions of cellular signalingpathways. After the treatment with δ-tocopherols inserted into themembranes, including the plasma membrane as well as the membranes inintracellular organelles, the membrane lipid movement is slightlyenhanced accounting for the slight reduction of free cholesterol in wildtype cells caused by an increase in cholesterol efflux. The resultantreduction in cholesterol esters is probably due to the increasing needfor free cholesterol. In NPC cells, the mutations in NPC1 and NPC2proteins result in the cessation of cholesterol movement in the membranesystem as well as the other lipids. The cholesterol efflux issignificantly reduced and free cholesterol accumulates in lysosome. Withδ-tocopherol treatment, the accumulated free cholesterols in lateendodome/lysosome are reduced which are mediated by an alternativeNPC1/NPC2-independent pathway. This alternative lipid movement pathwayis stimulated/enhanced by insertion of δ-tocopherols into the membranethat led to an increase in cholesterol efflux. Cholesterol efflux vialipid rafts and/or other vesicle are candidates for this alternativelipid movement pathway. This model explains the effect of δ-tocopherolson the reduction of enlarged lysosome size in four types of LSDs as theenhanced lipid movement may clean the accumulated lipids in lateendosome and lysosome.

Free cholesterol accumulation in cells is a hallmark of NPC disease. TheAmplex red cholesterol assay was used to screen an approved drug libraryin the NPC patient-derived skin fibroblasts that have free cholesterolaccumulation in late endosome and lysosome. Surprisingly, we discloseδ-tocopherol. δ-tocopherol significantly reduced the cholesterol amountin the NPCD fibroblasts. This cholesterol reduction effect wasconcentration dependent and proportional to the time of compoundtreatment (2 to 5 days) to the cells (FIG. 1 a). We then showδ-tocopherol in the filipin cholesterol staining assay, a method usedclinically for NPCD diagnosis. We disclose the cholesterol reductioneffect of δ-tocopherol and free cholesterol accumulation in NPCD patientcells was significantly reduced (FIG. 1 b). Lysosome size is usuallyenlarged in the NPCD cells using a lysotracker staining assay. As shownin FIG. 1 c, the enlarged lysosome size in NPCD fibroblasts wasdecreased and shrunken to the size similar to the wild type cells. Theseeffects on free cholesterol amount, filipin cholesterol staining, andlysotracker staining observed for δ-tocopherol were more significant andpotent than any of the other vitamin E isoforms. The effects ofcholesterol reduction and lysosome size decrease by δ-tocopherol in theNPC fibroblasts required a compound treatment for 2 to 5 days with asignificant effect after the 4-5 day treatment. δ-tocopherol at 40 μMwas more potent than α-tocopherol at 80 μM.

We disclose δ-tocopherol in 9 additional fibroblast primary cellsderived from different NPCD patients using above assays to examine thepotential variability in the cells derived from different NPCD patients.No significant difference was found in these cells and the effects oncholesterol reduction and lysosome size decrease were confirmed in allof these patient cells. Similar to the initial experimental results, theeffect of δ-tocopherol was more potent than that of α-tocopherol in allthese cells.

In order to examine if the cholesterol reduction effect of tocopherolswas related to an antioxidant effect, we tested a potent antioxidantagent N-acetyl cysteine (NAc) in the same set of assays. We found thatNAc did not have any effect on the cholesterol reduction nor lysosomesize decrease (FIG. 2 c), indicating that antioxidant effect of vitaminE may not account for the effects we observed on these NPCD cells. Wealso found that the other lipid soluble and water soluble vitaminsincluding vitamin D, vitamin K, and vitamin C were not active in theseassays further teaching that the effects of δ-tocopherol on cholesterolreduction and lysosome size decrease were specific and were notgeneralized in other vitamins (FIG. 2 c). We disclose that thecompound's cytotoxicity could interfere with these cell-based assaysthat often appeared as the false positives for cholesterol reduction andlysosome size decrease. We show that the cytotoxicity of compounds withthe cell/nuclear dye co-staining methods and an ATP content assay inparallel with the cholesterol and lysotracker assays. Cytotoxicity wasnot present at the effective concentration of these δ-tocopherol (FIG. 2d). δ-tocopherol, at a concentration of 50 uM or less did not showsignificant cytotoxicity after up to a 5-day compound treatment. Wefound that the impure compound preparation in combination with too highof a concentration of tocopherols could lead to the significantcytotoxicity in both wild type and NPCD fibroblasts. Although thetocotrienol effect on these NPCD cells were observed, they were moretoxic in our assays and the additional compound purification (to 99%).

It has been reported that the inhibition of cholesterol de novosynthesis and increase in NPC protein expression might correct thephenotype in these NPCD cells. In addition, the over expression of Rab9or acid sphingomyelinase has been reported to reduce the cholesterolaccumulation in the NPCD cells. We found that neither the expression ofHMG-CoA reductase, a key enzyme in the cholesterol synthesis pathway,nor that of NPC-1 protein was changed by the δ-tocopherol treatment(FIG. 2 d). The activity of HMG-CoA reductase or acid sphingomyelinasewas not changed as well. The Rab9 and acid sphingomyelinase proteinlevels remained same in these cells after the treatment with theδ-tocopherol (FIG. 2 d). We therefore disclose that these proteins maynot be directly involved in the mechanism of action of the δ-tocopheroltreatment as it relates to cholesterol reduction and lysosome sizedecrease. Thus, we further disclose a pathway for lipidrecycling/membrane movement in the membrane system which is enhanced bythe δ-tocopherol treatment. Through this enhanced lipid recyclingpathway, the clearance of the accumulated free cholesterols in lysosomeoccurs resulting in the decrease in lysosome size in these NPCD cells.

Skin fibroblasts derived from patients with lysosomal storage diseasesare commonly used for clinic diagnosis. They are also readily availablefrom Coriell Cell Repository and can be passaged up to 20-30 times incell culture. Although not all the patient derived fibroblasts exhibitlysosomal storage of macromolecules, the lipid storage and enlargementof lysosome size are showed in fibroblasts from patients with Farber,Niemann-Pick type A, Wolman, and Tay Sachs disease. We found that theenlargement of lysosome size in these four types of fibroblasts can bemeasured by the lysotracker dye staining method, similarly to that usedin the cells obtained from patients with Niemann-Pick type C disease.These patient derived fibroblasts can be used as the disease models fordrug testing. Farber, Niemann-Pick Type A, Wolman and Tay Sachs diseasescaused accumulation of ceramide, sphingomyelin, cholesterol-ester andGM2 gangliosides accumulation in lysosomes, respectively. (See FIGS.8-23).

Four other types of patient derived skin fibroblasts were tested in thelysotracker assay that visualizing the acidic compartments inside cellsincluding late endosome and lysosome. We disclose that δ-tocopherolsignificantly reduced the size of enlarged lysosomes in all four celltypes including these of Niemann Pick type A, Wolman, Farber andTay-Sachs diseases (FIG. 3, see also FIGS. 8-23). These cell types adistinct lysosomal storage disorders with the enlarged lysosomes incells. Cholesterol esters instead of free cholesterols are accumulatedin lysosomes of Wolman disease due to the mutations of acid lipase.Niemann Pick type A disease is caused by deficiency in acidsphigomyelinase (ASM) activity which results in accumulation ofsphingomyelin in lysosomes. Ceramides, produced by ASM fromsphingomyelin, accumulated in lysosomes as a result of ceramidasedeficiency in Farber disease. Tay-Sachs is caused by the hexosaminidaseA deficiency and accumulation of GM2 lipids in lysosomes. The structuresof these lipids involving these four additional lysosomal storagediseases are different from free cholesterol. We disclose the use ofδ-tocopherol on the lysosome size decrease in all of these diseases. Wefurther disclose in an alternate embodiment, that δ-tocopherol insertsinto the membrane and facilitates the movement of accumulated lipids outof lysosome resulting in a decrease in the enlarged lysosome size inthese cells.

The direct measurements using a GC-MS method revealed that the freecholesterol was reduced in the NPC fibroblasts after δ-tocopheroltreatment in NPCD fibroblasts (FIG. 4 a). The effect of δ-tocopherol wasprofound in the free cholesterol level in NPCD cells was nearlyrecovered to the normal level as in the wild type cells (FIG. 4 a),while the effect of α-tocopherol was not statistically different due tothe high variation among the three test groups. The free cholesterollevel in normal cells was also reduced slightly after the treatment. Theamount of cholesterol ester in both NPCD and wild cells after thetreatments of both α- and δ-tocopherol was significantly reduced (FIG. 4b). The sphingomyelin amount in both cell types was not significantlychanged after the treatment (FIG. 4 c). We also show that thecholesterol efflux significantly increased after the treatments withδ-tocopherol in the baby hamster kidney cells (FIG. 4 d), indicating thefree cholesterols removed from late endosomes and lysosomes weretransferred out of cells via the increased cholesterol efflux. Thus, wedisclose δ-tocopherol facilitates the lipid movement in membrane. Thereduction of intracellular free cholesterol as the result of increasedcholesterol efflux by δ-tocopherol mobilizes the storage cholesterol orthe utilization of storage cholesterols is also enhanced byδ-tocopherol.

The lethality of NPCD is primarily caused by the neuronal degenerationthough the cholesterol accumulation is observed in many cell types. Weshow the cholesterol reduction effect of δ-tocopherol is present inhuman NPC neuronal cells, using the neuron-like cells differentiatedfrom the mesenchymal stem cells (MSC) derived that were obtained fromtwo NPCD patients. Both the NPCD patient derived MSC cell lines showedthe similar cholesterol accumulation as that in the NPCD fibroblasts.The phenotype of cholesterol accumulation was preserved in theseneuron-like cells after the differentiation as well as in the MSC cells(FIG. 5 a). A set of neuronal markers were used to examine both wildtype and NPCD cells after the differentiation including including GAD65(glutamic acid decarboxylase), galactocerebroside, neurofilament M, NeuN(neuronal nuclear antigen) and neuron-specific beta III tubulin. We showthat only neuron-specific beta III tubulin was positive in both cellsafter neuronal differentiation whereas none of matured markers werefound, indicating that these cells were neuron-like cells but notmatured neurons. In addition, only a small portion of cells weredifferentiated similarly between wild type and mutant cells.δ-tocopherol treatment showed significant reduction in cholesterolaccumulation as well as a decrease in lysosome size in these neuron-likeNPCD cells that were co-localized by the filipin staining with aneuronal marker (FIG. 5 b). δ-tocopherol effects on the reduction ofaccumulated free cholesterol and decrease in lysosome size in lateendosomes and lysosomes exist in the neuron-like NPCD cells and the NPCDMSC cells, as it was observed in the NPCD fibroblasts.

The gene expression pattern of NPCD cells after the treatment withδ-tocopherol is disclosed in comparison with the wild type fibroblasts.(Table 1)

Among the genes identified in Table 1, the expressions of LDLR and ABCA1genes significantly reduced in NPC cells after the δ-tocopheroltreatment. NPC1 mutant cells were noted for an upregulation of (1)cholesterol homeostasis (transport) genes, (2) genes involved inmembrane trafficking, (3) genes associated with calcium regulation, (4)genes involved in oxidative stress and (5) genes associated withAlzheimer's disease.

NPCD patient-derived fibroblasts have an altered expression of manygenes that regulate lipid and cholesterol homeostasis, including thosethat affect lipid and protein transport, and de novo synthesis ofsaturated lipids and cholesterol (Table 1).

In the NPCD fibroblasts, LDLR and ABCA1 were upregulated inducing moretransport of cholesterol into the cell. The upregulation of LDLR in NPCcells is not the result of reduced available free cholesterols in cellsalthough they are accumulated in late endosomes and lysosomes. ABCA1transport was reported as one of cholesterol efflux mechanisms. It mayalso transport cholesterols into cells dependent on the balance ofcholesterols in and out of cells. The treatment with δ-tocopherol inNPCD cells decreased expression of these two genes. In addition, theexpression of several lipid and cholesterol synthetic genes, such as SCDand HMGCR, were also decreased after the δ-tocopherol treatment in NPCDcells which may be due to a direct interaction with or decreasedexpression of sterol sensors in the cell like SREBPs and INSIG1,reflecting an overall reduction in endoplasmic reticulum stress.

Beta-cyclodextrin (cyclo) has been reported to reduce cholesterolaccumulation in NPC mouse and cat models as well as increase in lifespan in these models. Cyclodextrin is also a pharmaceutical excipientthat is used for formulation of hydrophobic drugs. The mechanism ofaction for cyclo to treat NPC is still unclear. It has been implicatedthat the cyclo induced endocytosis and/or exocytosis may contribute toits effect on the cholesterol reduction in NPC cells and animal models.However, high concentration (0.3 to 10 mM in vitro) and large dose (4g/Kg body weight) are need for the effect of cyclo on the NPC that maycause the side and toxic effects. We disclose the combination therapy ofδ-tocopherol and cyclodextrin in reduced concentrations.

While the invention has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the invention and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims.

EXAMPLES

The following example is provided for illustrative purposes only and isnot intended to limit the scope of the invention.

Example 1

Human skin fibroblast cell lines GM5657, GM56599 and GM3123 werepurchased from the Coriell Cell Repository (Caden, N.J.). Other humanskin fibroblast cell lines NPC19, NPC20, and NPC25 were established byForbes D. Porter. Cell growth media (DMEM), L-glutamine, FBS,penicillin/streptomycin, Hoechst and LysoTracker Red were purchased fromInvitrogen (Carlsbad, Calif.). Filipin, gamma-tocopherol (T1782, >96%purity) were obtained from Sigma-Aldrich (St. Louis, Mo.).Delta-tocopherol was purchased from Sigma-Aldrich and purified to 99% byAAA using a protocol co-developed by AAA and NCGC.Alpha-(cat#100008377), gamma-(cat#10008494), delta-(cat#10008513)tocotrienols were obtained from Cayman Chemical (Ann Arbor, Mich.) inethanol solutions. They were lyophilized and dissolved in DMSO.Beta-tocopherol (99% purity, cat #ASB-00020319-050) was purchased fromChromaDex (Irvine, Calif.).

The primary human skin fibroblasts were cultured in DMEM media(Invitrogen, cat#11995-040) supplemented with 10% FBS, 100 unit/mlpenicillin and 100 ug/ml streptomycin in a humidified incubator (5% CO2)at 37° C.

The adipose mesenchymal stem cells (HMSC.AD-100, HMSC-NPC22 andHMSC-NPC23) were isolated from two NPC patients as well as one heathindividual and provided by Celleng-tech (Coralville, Iowa). These cellswere maintained in MSC expansion media (Celleng-tech,cat#HMSC.E.Media-450) supplemented with 10% FBS 100 unit/ml penicillinand 100 ug/ml streptomycin at 5% CO₂, 37° C. For neural differentiation,cells were initially plated at 30% confluency (˜2500 cells/cm²) usingMSC expansion media. After 24 hours, MSC expansion media were withdrew.Cells were washed twice with Dulbecco's phosphate Buffer saline(invitrogen) and incubated with neural differentiation media(Celleng-tech, cat#NEU.D.Media-450) supplemented with 10% FBS, 100unit/ml penicillin and 100 ug/ml streptomycin at 5% CO₂, 37° C. foradditional 3 days.

A collection of 2,800 compounds enriched in approved drugs were set upinternally at NIH Chemical Genomics Center and dissolved in DMSOsolution as 10 mM stock solution. The compounds were serially diluted at1:2.236 ratio in DMSO in 384-well plates to yield fifteen concentrationsand then formatted into 1,536-well plates at 5 μl/well as the compoundsource plates. The concentrations of diluted compounds in the sourceplates ranged from 0.28 μM to 10 mM. In the screen experiments, 23 nl ofcompounds in DMSO solution was transferred to assay plates containing 5ul/well cell culture medium. The final concentrations of compounds inthe screening assay plates ranged from 1.32 nM to 46 μM.

The free cholesterol level in these cells was then measured by acholesterol oxidase coupled HRP/amplex red detection method with acommercial available kit (Invitrogen). The cells were seeded to black,tissue culture-treated 1536-well plates (Greiner Bio-One, Monroe, N.C.)at 800 cells/well in 5 μl medium by a Multidrop Combi dispenser (ThermoScientific, Waltham, Mass.) and cultured for 24 hours. The assay plateswere added with 23 nl/well of compound DMSO solution using a pintoolstation (Kalypsys, San Diego, Calif.) and cultured for 3 days. The1536-well assay plate was washed by a plate-inverted centrifugationmethod that removing medium by centrifuging plates upside down against astack of paper towel at 1000 rpm for 1 min followed by addition of 7ul/well PBS buffer, repeating for two times. The PBS buffer was addedwith a 45 degree angled dispenser (Kalypsys) or a Multi-Drop Combidispenser set at medium speed to reduce disturbance to the cells. Afterthe cell wash, 2.5 ul/well of detection mixture from the kit was addedfollowed by 1 hour incubation at 37° C. With the addition of thedetection mixture, the cells were lysed by the detergent containingbuffer and free cholesterols in cell lysate were oxidized by cholesteroloxidase to produce H₂O₂ which reacting with amplex red dye in thepresence of horseradish peroxidase to yield red fluorescence signal. AViewLux plate reader (PerkinElmer, Boston, Mass.) was used to measurethe fluorescent signal in assay plates with the excitation of 573 nm andemissions of 610 nm.

Filipin dye staining was used to determine the level of free cholesterolaccumulation in cells (27, 28). Cells were seeded at 1000 cells/well in100 ul medium in the black/clear bottom, tissue culture treated 96-wellplates (Greiner Bio-One, Monroe, N.C.) for culture overnight or in adesignated time. The cells were washed twice with the HBBS buffer andfixed with 100 ul/well 3.2% formaldehyde at room temperature for 30minutes. After three times of plate wash with PBS buffer, cells werestained with 50 ug/ml filipin, which was freshly-dissolved in DMSO at 10mg/ml and then diluted in PBS buffer. Samples were then washed twicewith PBS buffer and stored in 4° C. On the day of imaging, cells werestained with 2 uM of CellMask Red (Invitrogen, H32711) in PBS at roomtemperature for 1 h. Samples were viewed and photographed using a NikonEclipse Ti microscope with a 20× objective equipped with CCD camera.DAPI filter and TRITC filter were used to visualize filipin and CellMaskRed, respectively. The images were processed by Adobe Photoshop.

Lysotracker is fluorescent dye that stains acidic compartments in livecells and is well-retained after aldehyde fixation. We have optimized alysotracker staining assay to visualize the enlarged lysosome size inthe NPC skin fibroblasts by applying appropriate concentration oflysotracker dye in comparison with these normal fibroblast cells. Cellswere seeded at 8000 cells/well in 100 ul medium in a black/clear bottom,tissue culture treated plate and cultured overnight or in a dedicatedtime. The cells were live-stained with 100 u/well 50 nM LysotrackerRed-99 dye (Invitrogen) dissolved medium at 37° C. for 1 hr followed bytwo-times plate wash with PBS buffer using the plate-invertedcentrifugation method. The plate was then added with 100 ul/well 3.2%formaldehyde and 1 ug/ml Hoechst 33342 (Invitrogen) in PBS and incubatedat room temperature for 30 minutes. After three times of plate wash withPBS buffer, samples were stored at 4° C. until imaging. Samples werephotographed using an IN Cell Analyzer 1000 (GE Healthcare) with a 20×objective equipped with CCD camera. DAPI filter and TRITC filter wereused to visualize Hoechst and Lysotracker red, respectively. The imageswere processed by Adobe Photoshop.

To measure the cytotoxicity of compounds, an ATP content assay was usedwith a commercial assay kit (ATP-Lite, PerkinElmer). Cells were seededin white solid 1536-well plates and treated with compounds same as inthe cholesterol oxidase assay describe above. After 3-day compoundtreatment, 3 ul/well detection mixture (prepared according to themanufacturer's instructions) was added to each well and the plates wereincubated for 10 min at room temperature followed by a measurement usinga Viewlux plate reader in a luminescence mode. The buffer in thedetection mixture lyses cells and releases cellular ATP which reactswith luciferin in the presence of luciferase to produce light. Theluminescence signal reduces if the cells are killed by the compoundduring the 3-day treatment.

Cell pellets were collected from 100-mm dishes and washed once with 10ml PBS. Proteins were assayed using the BCA kit (Sigma-Aldrich) andlipids were extracted by modified Bligh and Dyer procedure (29) from thecells. Internal standards were added based on protein concentration thatincluded N12:0 sphingomyelin, D7-cholesterol, 17:0 cholesterol ester(Avanti Polar Lipids, Inc). A portion of the lipid extract was used fordetection of sphingomyelins using direct infusion mass spectrometricassay with lithium hydroxide for facilitating ionization and the restwas used to convert cholesterol and its ester into acetate byderivatization. The derivatization was performed by treating the driedcrude lipid extract with solutions of 1M acetic acid with 1M DMAP inchloroform and 1M EDC in chloroform at 50° C. for 2 hours. Thederivatized product was extracted with hexane and analyzed by directinfusion mass spectrometric assay with 5% ammonium hydroxide forfacilitating ionization.

A triple-quadrupole mass spectrometer equipped with an electrospray wasused for analysis of lipids in positive mode. Sphingomyelins,cholesterol and cholesterol esters were detected by neutral loss scan of213 (collision energy: 50 V), neutral loss scan of 77 (collision energy:14 V) and precursor ion scan of m/z 369 (collision energy: 14 V)respectively. Data processing of mass spectrometric analyses includingion peak selection, data transferring, peak intensity comparison andquantitation was conducted using self-programmed Microsoft Excel macros(30).

MSC cells were seeded on coverslips on day 0 in MSC expansion medium. Onday 1, cells were washed twice with warm PBS buffer and then incubatedwith MSC neuronal differentiation medium. On day 4, media were replacedwith fresh neuronal differentiation medium. On day 6, cells were fixedwith 3.2% paraformaldehyde in HBSS for 15 min. Immunocytochemistry forneuronal beta III tubulin and unesterified cholesterol (filipinstaining) was performed as described. The coverslips were examined y aXXX microscope. The following filter sets were used: for filipin,excitation 360/40 nM, emission filter 460/50 nM.

Five NPC fibroblast cell lines as well as five normal fibroblast celllines were treated with 40 μM delta-tocopherol for 5 days with a mediumchange and sully of fresh compound on 3^(rd) day. The total RNAs wasprepared as the standard method described previously 0. The microarraygene expression experiment was carried out in a core microarray facilityat NHGRI with a set of AAAAA chips (Affymetrix).

Partek Genomics Software Suite (Copyright, Partek Inc.) was used toperform analysis of microarray data. Affymetrix cel files were loadedinto the software using the RMA algorithm, which involved backgroundcorrection, quantile normalization, log-2 transformation, and medianpolish summarization. PCA and hierarchical clustering identified onesample as an outlier. This sample (sample number 12) was not consideredfor further analysis.

ANOVA was performed to identify the differentially expressed genes forthree comparisons: 1) NPC_DMSO vs WT_DMSO 2) NPC_tocopheol-treated vsNPC_DMSO-treated and 3) WT_tocopherol-treated vs WT_DMSO-treated. An FDRcut-off of 0.05 failed to identify any genes with differentialexpression. So, a p-value cut-off of 0.01 was applied with an aim tovalidate the true positives using RT-PCR.

Cell lysates were prepared as previously described (31) and proteins inlysates were quantified using Bicinchoninic Acid (BCA) Protein Assay Kit(Sigma-Aldrich). Samples were heated at 65 C for 10 min before resolvedby SDS-PAGE under reducing conditions. Proteins were transferred to PVDFmembranes (Bio-Rad Laboratory) using either Criterion Blotter (Bio-RadLaboratory) or iBlot dry blotting devices (Invitrogen). For NPC1 andHMG-CoA reductase detection, rabbit polyclonal antibodies against NPC1(Abcam, cat #ab36983, 1:2000) or HMG-CoA reductase (Millipore, cat#07-457, 1:1000) were used, respectively. A peroxidase-conjugated donkeyanti-rabbit IgG (Santa Cruz) was used as secondary antibody at a 1:500dilution.

The compound library screen data was analyzed using software developedinternally at NIH Chemical Genomics Center (32). Concentration-responsecurves analyzed and EC50/IC50s were calculated with the Prism® software(GraphPad, San Diego, Calif.).

Example 2

Patient derived primary skin fibroblasts have been used as diseasemodels for testing the effect of alpha- and delta-tocopherols.Fibroblasts from patients with Farber disease, Niemann Pick Type Adiease, Wolman diseases and Say Tachs disease were obtained from CoriellCell repository (Camden, N.J.) that accumulating ceramide,sphingomyelin, cholesteryl ester and GM2 gangliosides in ysosomes,respectively.

(1) Amplex-red cholesterol assay. This biochemical assay (Invitrogen)uses cholesterol oxidase and a HRP-Amplex-re reporting system to measurethe free cholesterol level in cells. With the addition of acid lipase tothis assay, that hydrolyzes cholesterol ester to free cholesterol, thelevel of total cholesterol in cells including the cholesterol ester canbe measured. This assay was used for detecting the total cholesterollevel of in fibroblasts from patients with Wolman disease.

(2) Lysotracker dye (Invitrogen) staining assay. Lysotracker dye stainsthe acidic compartment in cells. We use an optimal concentration oflysotracker dye that shows the enlarged lysosomes due to the storage oflipids in fibroblasts from patients with lysosomal storage diseasesincluding Farber, Wolman, Say tachs and Niemann Pick Type A diseases.Drug treatment can reduce the size of the enlarged lysosome as the lipidstorage is decreased.

(3) Nile-red dye staining assay. Nile red dye (Invitrogen) stains twogroups of lipids. At excitation1=460-480 nm and emission1=535 nm, itstains the non-polar lipids such as cholesterol ester and triglycerides.At excitation2=500-540 nm and emission2 590-615 nm it stains the totallipids including the polar lipids such as phospholipids.

(4). Intracellular calcium assay. We used a fluorescence calcium dye(Fluo-8, AAT Bioquest, CA) to measure the transit increase ofintracellular calcium stimulated by alpha-tocopherol anddelta-tocopherol. Cytosal calcium increase is a second messenger whichmediates a variety of cellular functions including exocytosis.

δ-tocopherol has significant effect on reduction of enlarged lysosomesize and storage of macromolecules in patient derived fibroblasts offive lysosmal storage diseases. The mechanism of action for δ-tocopherolis direct insertion into the lipid membrane in cells that facilitateslipid movement in membrane and discharge of accumulated lysosomalmacromolecules out of cells. Thus, δ-tocopherol can be used to treat alltypes of lysosomal storage diseases. We teach the combination therapyusing vitamin E and cyclodextrin has synergetic or additive effect thatcan reduce dosage and adverse effect as well as increase the efficacydue to different mechanism of action for two drugs.

Example 3

It is envisioned that δ-tocopherol will be used either alone or incombination with inhibitors of CYP4F2 for the treatment of lysosomalstorage disorders. Purified Delta-tocopherol purified topharmacologically acceptable levels for the treatment of humans would beperformed. Dosage curves would be examined for both cytotoxicity andefficacy, as would be understood by one with skill in the art. Patientscould have multiple dose treatments, for multiple days, depending onefficacy, toxicity and half life with regard to delta tocopherol.

TABLE 1 Genes differentially expressed in NPC fibroblasts, or withtocopherol treatment Toco Toco NPC vs WT treat treat Gene Classuntreated NPC WT LDLR Cholesterol trafficking 1.3 −2.0 −2.0 ABCA1Cholesterol trafficking 1.3 −2.0 −1.4 CPE Intracellular transport 2.7 NCNC SCD Lipid and cholesterol synthesis 2.1 −3.9 −3.5 FADS2 Lipid andcholesterol synthesis 1.2 −2.5 −2.0 ACACA Lipid and cholesterolsynthesis 1.1 −1.5 −1.5 HMGCR Lipid and cholesterol synthesis 1.8 −1.7−1.5 HMGCS1 Lipid and cholesterol synthesis 1.6 −1.9 −1.6 SC4MOL Lipidand cholesterol synthesis 2.3 −1.4 −1.7 DHCR7 Lipid and cholesterolsynthesis 1.3 −1.7 −1.6 INSIG1 Cholesterol homeostasis 1.6 −1.8 −1.6SREBF1 Cholesterol homeostasis −1.1 −1.4 −1.2 SREBF2 Cholesterolhomeostasis 1.1 −1.6 −1.6 MMP1 Inflammation 4.9 2.7 5.0 MMP3Inflammation 5.1 1.7 2.0 TNFAIP6 Inflammation 2.9 NC NC FRRS1 FerricIron reductase 2.9 1.1 −1.1 NOX4 Generation of ROS 2.7 −1.1 −1.7

The description of the present invention has been presented for purposesof illustration and description, but is not intended to be exhaustive orlimiting of the invention to the form disclosed. The scope of thepresent invention is limited only by the scope of the following claims.Many modifications and variations will be apparent to those of ordinaryskill in the art. The embodiment described and shown in the figures waschosen and described in order to best explain the principles of theinvention, the practical application, and to enable others of ordinaryskill in the art to understand the invention for various embodimentswith various modifications as are suited to the particular usecontemplated.

1.-21. (canceled)
 22. A method for treating lysosomal storage disordercomprising administering to a subject in need thereof a pharmaceuticallyeffective composition comprising δ tocopherol.
 23. The method of claim22, wherein the lysosomal storage disorder is selected from the groupconsisting of Niemann-Pick Type C disease, Niemann-Pick Type A disease,Wolman disease, Farber disease, and Tay Sachs disease.
 24. The method ofclaim 23, wherein the lysosomal storage disorder is Niemann-Pick Type CDisease.
 25. The method of claim 22, wherein the composition comprisescyclodextrin.
 26. The method of claim 22, wherein the compositionexcludes α tocopherol.
 27. The method of claim 22, wherein thecomposition is administered in a dosage resulting in a plasmaconcentration of δ tocopherol of 10 μM to 50 μM.
 28. The method of claim22, wherein the composition is administered in a dosage of less than1000 IU/kg per day of δ tocopherol.
 29. The method of claim 22, whereinthe routes of administering are selected from the group consisting oforal, topical, suppository, intravenous, intradermic, intragaster,intramuscular, and intraperitoneal administration.
 30. A method forreducing cholesterol accumulation in cells comprising administering to ahost in need thereof an effective amount of a composition comprising δtocopherol, wherein the composition does not contain α tocopherol. 31.The method of claim 30, wherein the routes of administering are selectedfrom the group consisting of oral, topical, suppository, intravenous,intradermic, intragaster, intramuscular, and intraperitonealadministration.
 32. The method of claim 30, wherein the composition isadministered in a dosage resulting in a plasma concentration of δtocopherol of 10 μM to 50 μM.
 33. The method of claim 30, wherein thecomposition is administered in a dosage of less than 1000 IU/kg per dayof δ tocopherol.
 34. The method of claim 30, wherein the cholesterolaccumulation is free cholesterol.
 35. The method of claim 30, whereinthe cholesterol accumulation is in lysosomes of the cells.
 36. Themethod of claim 30, wherein the host is human.
 37. The method of claim22, wherein the composition comprises at least one inhibitor of CYP4F2.38. The method of claim 37, wherein the inhibitor is selected from thegroup consisting of ketoconazole, sesame seeds, lignan sesamin, andlignan sesaminol.
 39. The method of claim 30, wherein the compositioncomprises cyclodextrin.
 40. The method of claim 39, wherein thecyclodextrin is 2-hydroxypropyl-beta-cyclodextrin.